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Home , Tumor marker

Cerebrospinal Fluid Placental in the Intracranial : Results of

Nobuo ONO, Hirofumi NAGANUMA*, Hiroshi K. INOUE and Masaru TAMURA

Department of Neurosurgery, Gunma University School of Medicine, Maebashi , Gunma; *Department of Neurosurgery , Yamanashi Medical School, Yamanashi

Abstract

The authors investigated the placental alkaline phosphatase (PALP) activity in cerebrospinal fluid (CSF) by enzyme-antigen immunoassay using polyclonal as a marker for intracranial ger minomas in 17 patients with germ cell tumors and 20 with other disorders . The detection limit of PALP activity was 0.072 optical density units equivalent to 5.9 ng⁄ml.Five of nine germinomas demonstrated high CSF PALP activities before treatment. These high PALP activities became un detectable following radiation therapy. The other tumors were small or had no CSF contact. CSF PALP activity is a useful tumor marker for pure germinomas.

Key words: , tumor marker, placental alkaline phosphatase, enzyme immunoassay, cerebrospinal fluid

Introduction spinal fluid (CSF) as a method for diagnosing intracranial germinomas. Intracranial germinomas are often histologically unverifiable because of the pineal location and Materials and Methods radiocurability. Several oncodevelopmental markers have been investigated to differentiate the subgroups Our series of patients with intracranial germ cell of intracranial germ cell tumors. Alpha fetoprotein tumors between 1987 and 1990 included 15 males and (AFP) has been used as a tumor marker for em two females with ages ranging from 9 to 28 years bryonal or yolk-sac carcinoma, human (mean 15 years). Seven tumors were pineal, six were chorionic gonadotropin (HCG) for choriocar suprasellar, and four were in the basal ganglia or cinoma, and (CEA) for thalamus. There were nine germinomas, five mixed mature .2'9 '3) Although increased HCG germ cell tumors, one teratoma, one yolk sac levels are sometimes associated with germinoma, tumor, and one . The diagnosis AFP and HCG are very reliable for yolk-sac differen was histologically verified in 11 cases. The other tiated tumors and , respectively. six diagnoses were based on characteristic clinico In addition to these markers, placental alkaline radiological features, tumor markers, and response phosphatase (PALP), normally produced during to irradiation. The CSF samples were obtained on pregnancy by third trimester placenta, has recently admission and every 2 weeks after starting irradia been studied as a tumor marker for germinoma.'o,'s> tion. Samples were immediately frozen and stored PALP is detectable by and at -80°C until assayed. other methods.2,'o,",15) Control CSF samples were obtained from patients Here, we report our investigation into the enzyme with various conditions, including 11 males and nine antigen immunoassay of PALP activity in cerebro females with ages ranging from 7 months to 66 years (mean 38 years). There were seven parkinsonisms, Received June 12, 1990; Accepted January 30, eight brain tumors (pineocytoma, pineal , 1991 , craniopharyngioma, pineal glial cyst, gliosarcoma, hypothalamic hamartoma, and subependymal giant cell ), one brain abscess, one inflammatory granuloma, one multiple sclerosis, one idiopathic diabetes insipidus, and one post-stroke pain. Ten CSF samples were obtained from ventricular taps during stereotactic operations. The other CSF samples were taken through lumbar taps or Ommaya reservoirs. The assay was performed by techniques described elsewhere with some modification.',",") Rabbit anti human immunoglobulin (The Green Cross Corp., Osaka) at 1 : 200 dilution was adsorbed in a 96-well immunoplate (Greiner, Nurtingen, Germany) by overnight incubation at 4°C (50,u1/well). Unbound antibody was then removed by washing with phosphate-buffered saline (PBS). PBS containing gelatin 10 gm/1 was adsorbed in the plate wells. The plate was washed again with PBS, before 50,ul CSF sample was dispensed into each well. All samples Fig. 1 Dilution curves of standard PALP after 2 were assayed in duplicate or triplicate. The plates hours (•) and overnight (A) incubation. were incubated for 1 hour at room temperature in Values are means ± SD for 6 separate assays. cluding heat-treatment for 20 minutes at 65'C. The enzyme activity was then determined colori metrically by adding 50,ul substrate solution to the wells, incubating at 37°C for 2 hours and then standing overnight (18-24 hours) to develop the color reaction. The optical density (OD) at 405 nm of the plate wells was determined using a Model M580 spectrophotometer (Microelisa Autoreader, Dynatech Products). Single or repeated assays were performed.

Results

Figure 1 shows the log OD405 against enzyme activity curves for dilutions of PALP standard samples (P3895, Sigma Chemical Co., St. Louis, Mo., U.S.A.) after 2 hours and overnight incubation, resulting from 6 separate assays of the same PALP standards over 1 month. The internal assay accuracy Fig. 2 Changes in pre and post-treatment CSF PALP was tested using different plates in the same assay activity in intracranial germinoma cases (n = and parallel measurements of 3 dilution curves. 9). The mean coefficient of variation (CV) was 9.9%. The accuracy between assays was tested on 6 differ freezing/thawing, did not affect the immunological ent days. The mean interassay CV was 16.6%. The PALP determination, but repeated freezing and detection limit for PALP was 0.072 OD units equiv thawing decreased the PALP activity. Samples to be alent to 5.9 ng/ml after overnight incubation. OD stored for longer than 1 week were stored at -20°C values lower than 0.072 had a mean CV of more or -80°C. PALP activity decreased more when than 20%. The background absorbance in these samples were stored at -20°C than at -80°C. assays was lower than 0.023 OD for wells (n = 20) The control group demonstrated a mean value of in which PBS was substituted for the test samples. 0.028 OD. In contrast, five of nine germinoma The influence of storage at 4°, -20°, and -80°C patients demonstrated pretreatment CSF PALP on the results of the assay was determined. Storage at activities exceeding 0.095 OD. Marked reductions 4°C for not longer than 1 week, or 1 cycle of occurred in the 2 weeks after initiating irradiation (Fig. 2). Pre and post-treatment PALP activities in the eight patients with other germ cell tumors were lower than 0.044 OD. Pretreatment CSF samples from the nine ger minoma patients were also assayed for HCG, AFP, and CEA. A higher CSF PALP activity was not significantly associated with either AFP, HCG, ,8 HCG, or CEA. Three of nine germinoma patients produced HCG or 8-HCG, demonstrating 2554 mIU/ml, 7200 mIU/ml, and 3.6 ng/ml. All three tumors were biopsied. The former two cases were ger Fig. 4 MR images with Gd-DTPA in Case 2. left: minomas with syncytiotrophoblastic giant cells and Pretreatment MR image showing enhanced the other was a germinoma. One patient produced masses in pineal and sellar regions. right: Post-treatment MR image revealing no tumor AFP, at a serum level of 17 ng/ml (Case 1). evidence.

Representative Case Reports

Case 1: A 14-year-old male was admitted with a 1 year history of polydipsia and polyuria on February 13, 1987. A computed tomographic (CT) scan show ed no evidence of a tumor. He was initially treated for idiopathic diabetes insipidus. One year later, a repeat postcontrast CT scan disclosed a sellar mass (Fig. 3 left). On admission, he also complained of rapid fatigue and cold intolerance. Neurological examination showed no deficit. AFP, HCG, and CEA CSF tumor markers and cytological studies were all negative. However, the levels of serum AFP and CSF Fig. 5 Case 2. Changes in CSF PALP activity PALP were 17 ng/ml and 0.174 OD, respectively. A following treatment. diagnosis of germinoma with a possible malignant component was made. No was made. He received irradiation (whole brain 34 Gy, booster 16 Case 2: A 15-year-old male was referred to our clinic Gy, and spine 30 Gy). A post-treatment postcontrast on January 26, 1990. His weight had decreased by 5 CT scan 6 weeks later showed no tumor (Fig. 3 right). kg since July, 1989, when he complained of Serum AFP became undetectable and the CSF headache, thirst, and rapid fatigue, accompanied by PALP level had decreased to 0.032 OD. a decrease in pubic and axillary hair. Neurological and endocrinological studies showed Argyll-Robert son pupils and panhypopituitarism. A T,-weighted magnetic resonance (MR) image with gadolinium diethylene-triamine-pentaacetic acid (Gd-DTPA) disclosed enhanced masses in the pineal and sellar regions (Fig. 4 left). The CSF PALP activity was 1.010 OD. The cytological study was compatible with germinoma, although serum and CSF AFP, HCG, and CEA markers were all undetectable. Following irradiation (whole brain 30 Gy, booster 20 Gy, and spine 30 Gy), a MR image showed no tumor evidence (Fig. 4 right) and the CSF PALP activity was decreased to 0.040 OD (Fig. 5). Fig. 3 Postcontrast CT scans in Case 1. left: Pre treatment CT scan disclosing a sellar mass. Discussion right: Post-treatment CT scan demonstrating no tumor. The serological PALP assay was originally in vestigated to monitor testicular germ cell not be uniform. 12)This assay may then be useful for tumors, 1,1,1,1,16,11)because active patients had estimating the relative proportion of germinoma pre increased serum PALP levels. Polyclonal antisera sent in patients. However, since the enzyme activity and then monoclonal antisera were used to demon in the sample decreases with time, this method must strate that seminoma expressed PALP. However, be regarded as qualitative, not quantitative. We now monoclonal , which have a high selectivity try to determine the PALP activity as soon as the for a specific PALP, may not recognize some sample is obtained. infrequent alkaline forms of PALP.6) Hofmann Despite these disadvantages, our laboratory uses et al.'s reported that could express both the PALP CSF assay in suspected placental alkaline phosphatase and germ cell alka cases since intracranial germinomas may be treated line phosphatase, which are similar, but can be sepa by irradiation without biopsy. Germinoma is rated by immunoelectrophoresis. As germinomas sometimes very difficult to differentiate from other possibly produce one or both isoenzymes, it is neces radiosensitive brain tumors such as pineoblastoma, sary to use a polyclonal antibody to avoid a false although the majority of pineal tumors are agreed to negative result. be of germinal origin. An enzyme-linked immunosorbent assay showed some cross-reactivity between the polyclonal anti Acknowledgment body and intestinal alkaline phosphatase,') because antibodies against PALP share antigenic deter We express our appreciation to Professor James A. minants with the intestinal isoenzyme. Both isoen Taren, Department of Neurosurgery, University of zymes are part of the total activity of serum alkaline Michigan Medical Center, U.S.A., for his advice and phosphatase. Serum intestinal alkaline phosphatase, reviewing this manuscript. which increases after a fatty meal, has been identified not only in patients with liver cirrhosis, but also in normal people with type 0 or B.8 Smoking References enhances PALP-like alkaline phosphatase synthesis 1) Hofmann MC, Jeltsch W, Brecher J, Walt H: Alka and secretion by the lung alveoli, and can also in line phosphatase isozymes in human testicular germ fluence the PALP expression. cell tumors, their precancerous stage, and three To avoid the false positive results in serological related cell lines. Res 49: 4696-4700, 1989 PALP assays as described above, PALP activities 2) Inoue HK, Naganuma H, Ono N: Pathobiology of were measured using CSF, where alkaline intracranial germ-cell tumors: Immunochemical, phosphatase is naturally absent. Pretreatment CSF immunohistochemical, and electron microscopic in PALP activities were higher in germinoma patients, vestigations. J Neurooncol 5: 105-115, 1987 but undetectable in other germ cell tumors and 3) Jemmerson R, Fishman WH: Convenient selection of posttreatment germinomas. The enzyme-antigen im monoclonal antibodies to isoenzymes of placental munoassay used a relatively simple method, which re alkaline phosphatase using the catalytic activity of the antigen in enzyme-antigen immunoassay. Anal quired none of the special precautions and/or separa Biochem 124: 286-292, 1982 tion steps necessary for assays using radioactive 4) Jeppsson A, Wahren B, Stigbrand T, Edsmyr F , materials or anti-PALP horseradish-peroxidase con Andersson L: A clinical evaluation of serum placen jugate. The simplicity, sensitivity and specificity of tal alkaline phosphatase in seminoma patients. Br J this assay make it a useful method for measuring Urol 55: 73-78, 1983 PALP in pure germinomas, which do not produce 5) Lange PH, Millan JL, Stigbrand T, Vessella RL, AFP, HCG, or CEA, and also make individual Ruoslahti E, Fishman WH: Placental alkaline testing more cost effective. phosphatase as a tumor marker for seminoma. However, CSF PALP testing was negative in four Cancer Res 42: 3244-3247, 1982 of the nine pretreatment germinomas. These tumors 6) McLaughlin PJ, Gee H, Johnson PM: Placental-type were small, less than 2 cm in diameter, or situated in alkaline phosphatase in pregnancy and malignancy the basal ganglia without direct CSF contact. plasma: Specific estimation using a monoclonal antibody in a solid phase enzyme immunoassay. Therefore, measurement of the CSF PALP activity is Clin Chim Acta 130: 199-209, 1983 valuable if the tumor is located in the ventricular 7) Millan JL, Stigbrand T: “Sandwich”enzyme im walls and has broad contact with the CSF. The munoassay for placental alkaline phosphatase. Clin simultaneous measurement of AFP, HCG, CEA, Chem 27: 2014-2018, 1981 and PALP is necessary for accurate monitoring, 8) MössnerE, Pfleiderer G: Solid-phase direct im since the composition of an individual tumor may munoassay for serum intestinal and placental alkaline phosphatase. Clin Biochem 16: 28-30, 1983 munoassay for human placental alkaline phosphatase 9) Naganuma H, Inoue HK, Nakamura M, Koizumi H: in serum and tissue extracts, and its application as a Localization of carcinoembryonic antigen in mature tumor marker. Clin Chem 31: 41-45, 1985 intracranial . J Neurosurg 62: 870-873, 15) Shinoda J, Yamada H, Sakai N, Ando T, Hirata T, 1985 Miwa Y: Placental alkaline phosphatase as a tumor 10) Ono N, Inoue HK: Primary intracranial germ cell marker for primary intracranial germinoma. J tumors. Kita Kanto Igaku 39: 657-671, 1989 (in Neurosurg 68: 710-720, 1988 Japanese) 16) Tucker DF, Oliver RTD, Travers P, Bodmer WF: 11) Ono N, Inoue HK, Naganuma H, Kunimine H, Serum marker potential of placental alkaline Zama A, Tamura M: Diagnosis of germinal phosphatase-like activity in testicular germ cell neoplasm in the thalamus and basal ganglia. Surg tumours evaluated by H 17E2 monoclonal antibody Neurol 26: 24-28, 1986 assay. Br J Cancer 51: 631-639, 1985 12) Ono N, Inoue HK, Naganuma H, Misumi S, Tamura 17) Wahren B, Holmgren PA, Stigbrand T: Placental M: Germ cell tumor in the basal ganglia: Im alkaline phosphatase, alpha fetoprotein and car munohistochemical demonstration of a-fetoprotein, cinoembryonic antigen in testicular tumors. Tissue human chorionic gonadotropin, and carcinoem typing by means of cytologic smears. Int J Cancer 24: bryonic antigen. Surg Neurol 25: 495-500, 1986 749-753, 1979 13) Ono N, Takeda F, Uki J, Zama A, Hayashi Y, Sampi K: A suprasellar producing alpha-fetoprotein and human chorionic gonadotro pin: Treated with combined chemotherapy followed Address reprint requests to: N. Ono, M.D., Department of by radiotherapy. Surg Neurol 18: 435-443, 1982 Neurosurgery, Gunma University School of 14) Pollet DE, Nouwen EJ, Schelstraete JB, Renard J, Medicine, 3-39-22 Showa-machi, Maebashi, Gunma Vande Voorde A, De Broe ME: Enzyme-antigen im 371, Japan.